The occurrence of serious bacterial and fungal infections is an increasing problem despite notable progress in antibiotic therapy. Each year there are more than 40 million hospitalizations in the United States of America and more than 2 million of these patients become infected in the hospital. Antibiotic-resistant bacteria are involved in 50-60% of these cases (Tomasz A. Multiple-Antibiotic-Resistant Pathogenic Bacteria—A Report on the Rockefeller-University Workshop. New England Journal of Medicine 330: 1247-51, 1994). These hospital-acquired diseases are estimated to lead to 60 000 to 70 000 deaths in the USA and up to 10 000 deaths in Germany (Wenzel R P. The Mortality of Hospital-Acquired Blood-Stream Infections—Need for A New Vital Statistic. International Journal of Epidemiology 17: 225-7, 1988). Whereas resistant Gram-negative bacteria were the main problem in the 1970s, in the last decade there has been an increase in cases in which Gram-positive bacteria that are resistant to several antibiotics play a role (Moellering R C. Emerging resistance with gram-positive aerobic infections: Where do we go from here? Introduction: Problems with antimicrobial resistance in gram-positive cocci. Clinical Infectious Diseases 26: 1177-8, 1998). The current rapid development of resistant strains involves both Gram-positive and Gram-negative pathogens (Hand W L. Current challenges in antibiotic resistance. Adolescent Medicine 11: 427-38, 2000). Resistances developed first in species in which single mutations were sufficient to reach clinically important levels, e.g. Staphylococcus aureus and Pseudomonas aeruginosa; next were bacteria in which multiple mutations were necessary, for instance E. coli and Neisseria gonorrhoeae. This is due primarily to the frequent use of fluoroquinolone antibiotics (Hooper D C. Emerging mechanisms of fluoroquinolone resistance. Emerging Infectious Diseases 7: 337-41, 2001). Another important cause of the development of resistance in Gram-negative bacteria is the extensive range of lactamases in Escherichia coli and Klebsiella pneumoniae (Jones R N. Resistance patterns among nosocomial pathogens—Trends over the past few years. Chest 119: 397S-404S, 2001). Nearly half the clinically relevant strains of Haemophilus ducreyi, the causative agent of soft chancre, carry genes that make this bacterium resistant to amoxicillin, ampicillin and several other β-lactams (Prachayasittikul V, Lawung R, & Bulow L. Episome profiles and mobilizable beta-lactamase plasmid in Haemophilus ducreyi. Southeast Asian J Trop Med Public Health 31: 80-4, 2000). Similarly, the resistance of Salmonella enterica serovar typhimurium to tetracyclines rose from zero percent in the year 1948 to 98% in the year 1998 (Teuber M. Spread of antibiotic resistance with food-borne pathogens. Cellular and Molecular Life Sciences 56: 755-63, 1999).
This explains the need for further searching for new antibiotics. Inducible antibacterial peptides represent a field of research in which modern biochemistry, immunology and research into active substances come together. Peptide antibiotics, ranging in size from 13 to more than a hundred amino acids, have been isolated from plants, animals and microbes (Boman H G. Peptide Antibiotics and Their Role in Innate Immunity. Annual Review of Immunology 13: 61-92, 1995). A single animal has approx. 6-10 peptide antibiotics, with each peptide often displaying a completely different activity spectrum (Barra D, Simmaco M, & Boman H G. Gene-encoded peptide; antibiotics and innate immunity. Do ‘animalcules’ have defense budgets? Febs Letters 430: 130-4, 1998). It is known that the overwhelming number of antibacterial peptides, including the much-studied defensins, cecropins and magainins, act by a “lytic/ionic” mechanism. A permeabilizing effect on the bacterial cytoplasmic membrane has been discussed as a common mechanism of action of these “lytic” peptides (Ludtke S, He K, & Huang H. Membrane thinning caused by magainin 2. Biochemistry 34: 16764-9, 1995; Wimley W C, Selsted M E, & White S H. Interactions Between Human Defensins and Lipid Bilayers—Evidence for Formation of Multimeric Pores. Protein Science 3: 1362-73, 1994; Shai Y. Molecular Recognition Between Membrane-Spanning Polypeptides. Trends in Biochemical Sciences 20: 460-4, 1995). A cationic, amphipathic structure, which forms hydrophilic ion (proton) channels in a lipid bilayer, is the basis of this activity. Owing to the outflow of protons, the membrane potential that is necessary for many fundamental life processes is disturbed and as a result the cell is killed. Since disturbance of the membrane by these peptides is dependent on the recognition of chiral molecules, an amino acid exchange, which does not remove the general amphipathic structure or basic net charge, is tolerated functionally (Wade D et al. All-D Amino Acid-Containing Channel-Forming Antibiotic Peptides. Proceedings of the National Academy of Sciences of the United States of America 87: 4761-5, 1990; Steiner H, Andreu D, & Merrifield R B. Binding and Action of Cecropin and Cecropin Analogs—Antibacterial Peptides from Insects. Biochimica et Biophysica Acta 939: 260-6, 1988). At higher concentrations these lytic peptides often have toxic action on mammalian membranes, which limits their suitability as possible medicinal products. If proline is inserted into the sequence of the α-helical antimicrobial peptides, the capacity of the peptides to permeabilize the cytoplasmic membrane of E. coli decreases as a function of the number of proline residues. On examining this, it is amazing that some of the most active, native antibacterial peptides, at least with respect to some Gram-negative pathogens, belong to the family of proline-rich peptides (Otvos L et al. Insect peptides with improved protease-resistance protect mice against bacterial infection. Protein Science 9: 742-9, 2000).
The side effects described above are overcome by antimicrobial peptides (AMP), which specifically recognize a bacterial protein or other intra- or extracellular components, without displaying cross-reactivity with mammalian analogs. This seems to apply to proline-rich antimicrobial peptides, including apidaecins, drosocin and pyrrhocoricin which were originally isolated from insects. With the enormous variation in size and biochemical properties, it is not surprising that the structure-activity and conformation-activity relations are the focus of antibacterial peptide research. A complete investigation of the natural antibacterial peptide repertoire for biological strength is important not only for general biochemical questions, but is also of constant interest for the pharmaceutical industry. Despite the problems of in-vitro tests with peptide-based antibiotics, some natural, cationic antibacterial peptides have already reached the clinical trial phase (Boman H G. Peptide Antibiotics and Their Role in Innate Immunity. Annual Review of Immunology 13: 61-92, 1995). Whereas some of these peptides showed activity as topical (local) agents in the early clinical trial phase, others were active in systemic therapy. For example, the cationic protein rBPI 21, which is used for parental treatment of meningococcemia, has completed the third phase of clinical testing (Boman H G. Peptide Antibiotics and Their Role in Innate Immunity. Annual Review of Immunology 13: 61-92, 1995).
The family of the proline-rich peptides (e.g. apidaecin, drosocin and pyrrhocoricin) kill bacteria not only by permeabilization of their membrane, but bind stereospecifically to one or more target proteins. These possible interaction partners, up to now the heat-shock protein DnaK has been investigated thoroughly (Kragol G et al. Identification of crucial residues for the antibacterial activity of the proline-rich peptide, pyrrhocoricin. European Journal of Biochemistry 269: 4226-37, 2002; Kragol G et al. The antibacterial peptide pyrrhocoricin inhibits the ATPase actions of DnaK and prevents chaperone-assisted protein folding. Biochemistry 40: 3016-26, 2001), are inhibited by the proline-rich peptides and presumably the correct protein folding is prevented, ultimately leading to cell death. Moreover, proline-rich peptides, in stark contrast to AMPs with defined secondary structure such as melittin or gramicidin, seem in vitro to have neither hemolytic nor toxic effects on eukaryotic cells. Along with antimicrobial activity, mainly the stability in mammalian serum (25%) has a decisive influence on the development of new peptide-based antibiotics. For example, drosocin is broken down within an hour, whereas pyrrhocoricin is far more stable with respect to proteases, with half-lives of 120 minutes.
In biological experiments by Schneider and Dorn (2001) (Schneider M & Dorn A. Differential infectivity of two pseudomonas species and the immune response in the milkweed bug, Oncopeltus fasciatus (Insecta: Hemiptera). Journal of Invertebrate Pathology 78: 135-40, 2001), nymphs and pupae of the milkweed bug Oncopeltus fasciatus from the Lygaeidae family were infected with two different Gram-negative Pseudomonas species and their immune response was analyzed. Whereas infection of the nymphs of O. fasciatus with the human pathogen Pseudomonas aeruginosa resulted in the death of all individuals after 48 h, 71% of individuals infected with the less pathogenic Pseudomonas putida survived for at least 96 h. If the nymphs of the milkweed bug were then infected first with P. putida and after 24 h with P. aeruginosa, the survival rate of the doubly infected individuals within the first 24 h rose significantly to 73%. The probable induction of synthesis of antibacterial peptides, by which insects defend themselves, within the scope of their innate immune system, against invading microorganisms, was then investigated. Four peptides (Oncopeltus antibacterial peptide 1-4) were identified with molecular weights of 15, 8, 5 or 2 kDa and were held to be responsible for the antibacterial action. Sequence analysis according to Edman found, in addition to a 34 amino acid long partial sequence for peptide 1 (15 kDa), also the incomplete sequence of the proline-rich 2 kDa peptide 4. The amino acids in positions 11 and the C-terminal sequence starting from position 19 could not be identified definitively. The exact molecular weight is unknown.
A selection of currently known sequences of antibiotic peptides is presented in Table 1:
TABLE 1SEQ IDPeptideSpeciesSequenceNO.Ref.Apidaecin 1aApisGNNRPVYIPQPRPPHPRI119[1]mellifera Apidaecin 1bApisGNNRPVYIPQPRPPHPRL87[1]mellifera DrosocinDrosophilaGKPRPYSPRPTSHPRPIRV89[2]melanogaster Formaecin 1MyrmeciaGRPNPVNNKPTPYPHL120[3]gulosa PyrrhocoricinPyrrhocorisVDKGSYLPRPTPPRPIYNRN-NH291[4]apterus Metalnikowin 1PalomenaVDKPDYRPRPRPPNM121[5]prasina OncopeltusOncopeltusEVSLKGEGGSNKGFIQGSGTKTLFQDD122[6]antibacterialfasciatusKTKLDGTpeptide 1 OncopeltusOncopeltusVDKPPYLPRP(X/P)PPRRIYN(NR)123[6]antibacterialfasciatuspeptide 4 [1] Casteels P, Ampe C, Jacobs F, Vaeck M, & Tempst P. Apidaecins - Antibacterial Peptides from Honeybees. Embo Journal 8: 2387-91, 1989[2] Bulet P et al. A Novel Inducible Antibacterial Peptide of Drosophila Carries an O-Glycosylated Substitution. Journal of Biological Chemistry 268: 14893-7, 1993[3] Mackintosh J A et al. Isolation from an ant Myrmecia gulosa of two inducible O-glycosylated proline-rich antibacterial peptides. Journal of Biological Chemistry 273: 6139-41, 1998[4] Cociancich S et al. Novel Inducible Antibacterial Peptides from A Hemipteran Insect, the Sap-Sucking Bug Pyrrhocoris-Apterus. Biochemical Journal 300: 567-75, 1994[5] Chernysh S, Cociancich S, Briand J P, Hetru C, & Bulet P. The inducible antibacterial peptides of the hemipteran insect Palomena prasina: Identification of a unique family of proline-rich peptides and of a novel insect defensin. Journal of Insect Physiology 42: 81-9, 1996[6] Schneider M & Dorn A. Differential infectivity of two pseudomonas species and the immune response in the milkweed bug, Oncopeltus fasciatus (Insecta: Hemiptera). Journal of Invertebrate Pathology 78: 135-40, 2001
There is still a demand for new antibacterial and antimycotic compounds, new antibacterial and antimycotic pharmaceutical compositions, as well as methods using them, and compounds that can be used for screening active substances, to detect new pharmaceutical antibiotics.
The problem to be solved by the present invention is to provide new antibiotic peptides with increased stability, to extend the spectrum of action of the AMPs on Gram-positive bacteria and thus make modern broad-spectrum antibiotics available, and to introduce the peptides into eukaryotic cells and thus combat hidden bacteria.